Differential solubility and subunit composition of rat glomerular basement membrane.
Glomerular basement membrane (GBM) was prepared from 2- to 3-month-old Sprague-Dawley rats by differential sieving and sonication. 80% of the membrane was soluble in 1% sodium dodecyl sulphate (SDS) and 1% 2-mercaptoethanol. The soluble fraction was resolved into 15 bands in the molecular weight range 30,000 to 300,000 by SDS-polyacrylamide gel electrophoresis. The major bands present had apparent molecular weights of 100,000 and 148,000. Treatment of GBM with SDS alone solubilised mainly low molecular weight components (45,000-150,000) but when the residue was treated with SDS and 2-mercaptoethanol higher molecular weight material was solubilised. Partial solubilisation of GBM was also achieved with pepsin. Digestion for 18 h at 4 degrees C resulted in 20% of the membrane being solubilised but this was increased to 55% at 10 degrees C. The amino-acid composition of pepsin-soluble GBM was more collagen-like than the residue remaining after enzyme digestion. Although the residue was more polar than whole GBM it still contained significant amounts of glycine and hydroxyproline and could be further subfractionated with SDS into a soluble fraction, the amino acid content of which was similar to whole GBM and a collagenous residue containing 317 residues/1,000 of glycine. When pepsin solubilised GBM was subjected to horizontal electrophoresis in SDS-polyacrylamide slab gels the principal bands migrated in the pro-alpha-chain region. This material was heterogeneous and in addition to the principal components, 6 components with apparent molecular weights less than 95,000 were present together with high molecular weight material in the gamma and beta regions of the gel. The band pattern of the pepsin-insoluble material was similar although the intensities of some individual bands varied significantly from that of the pepsin-soluble material. Rat GBM can therefore be fractionated by treatment with SDS alone, SDS together with 2-mercaptoethanol and pepsin digestion. The data reported is compatible with the presence of a mixture of collagen-like and polar regions rather than a major single collagenous component (type IV collagen).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001485:Basement Membrane / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D003094:Collagen / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007678:Kidney Glomerulus / Q000032:analysis*; D008970:Molecular Weight; D010434:Pepsin A; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D000596:Amino Acids; D003094:Collagen; D010434:Pepsin A